The goal of this revised proposal is to characterize vertebrate genes that play a role in neuromuscular synapse formation and maintenance, using zebrafish as a model system. We participated in a small-scale mutagenesis screen conducted by Drs. Mary Mullins and Michael Granato in the Dept. of Cell and Developmental Biology at the University of Pennsylvania. Using antibodies against synaptic vesicles to label presynaptic terminals, fluorescently conjugated abungarotoxin to label acetylcholine receptor (AChR) clusters, and light microscopy in intact fish, my lab identified eight mutants with defects in different aspects of neuromuscular synaptogenesis at 48 hours post fertilization (hpf). These include mutants with reduced pre- and/or postsynaptic specializations, abnormal size, alignment or clustering of pre- or postsynaptic specializations, and aberrant primary and/or secondary motor axon branching within the body wall musculature, resulting in aberrant synaptic localization. Many of these mutant phenotypes have not been previously reported in zebrafish and are distinct from those described in other organisms. Here I propose to: 1) Study neuromuscular synaptogenesis using in vivo imaging in wild type and transgenic fish in which presynaptic axons and terminals are labeled with GFP, and other approaches; 2) Determine the cellular and molecular phenotype of bossu, pongo, slytherin and xavier; 3) Determine a fine map location for bossu, xavier, pongo and slytherin; and 4) Determine the identity of the mutant gene in bossu and xavier. Together, these approaches will allow us to study the genetic, molecular and cellular mechanisms underlying synaptogenesis in vertebrates. Identification of the underlying genetic defects in these mutants will expand our understanding of the molecular mechanisms that mediate the formation and function of neuromuscular and other synapses.